Novel viral particles for therapeutic purposes

Abstract

The present invention relates to viral particles comprising a Lymphocytic Choriomeningitis Virus (LCMV) S segment and an LCMV L segment, where the S segment comprises an open reading frame encoding a glycoprotein derived from LCMV strain WE, and the L segment comprises an open reading frame encoding an L protein derived from LCMV strain clone 13. The invention also relates to associated host cells, methods of producing such viral particles, pharmaceutical compositions comprising such viral particles, and medical uses of such viral particles.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to European patent application EP 21159746.3, filed February 26, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THEINVENTION The present invention relates to viral particles comprising a Lymphocytic Choriomeningitis Virus (LCMV) S segment and an LCMV L segment, where the S segment comprises an open reading frame encoding a glycoprotein derived from LCMV strain WE, and the L segment comprises an open reading frame encoding an L protein derived from LCMV strain clone 13. The invention also relates to associated host cells, methods of producing such viral particles, pharmaceutical compositions comprising such viral particles, and medical uses of such viral particles. [Background technology]

[0003] background LCMV belongs to the genus Mammarenavirus and can be ubiquitously isolated from mice and other rodents (Radoshitzky et al. (2020) in DM Knipe and PM Howley (eds.), Fields Virology (Non-Patent Document 1)). Intrauterine infection leads to persistent infection of offspring, with potentially very high viral loads but no clinical symptoms (Radoshitzky et al. (2020) in DM Knipe and PM Howley (eds.), Fields Virology (Non-Patent Document 1)). Live virus is shed from infected animals throughout their lives via urine, saliva, nasal secretions and feces, causing symptomatic infection in exposed naive rodents.

[0004] Humans are infected with LCMV by close contact with infected rodents or secretions from infected rodents, by solid organ transplantation, or by vertical transplacental transmission. Horizontal human-to-human transmission outside of solid organ transplantation has not been reported. Postnatal infection in individuals with intact immune systems is often asymptomatic or causes a mildly febrile illness. Mild, reversible neurological symptoms are rarely observed. Infection during pregnancy increases the risk of miscarriage, severe CNS, or ocular malformations. Neurological sequelae include spastic quadriplegia, mental retardation, seizures, and visual impairment. In immunosuppressed organ recipients, LCMV infection can be fatal, although fewer than 20 cases have been reported to date.

[0005] LCMV strains are divided into four different lineages, with the most commonly used laboratory strains Armstrong and WE grouped into lineage I (Radoshitzky et al. (2020) in DM Knipe and PM Howley (eds.), Fields Virology). These laboratory strains have low pathogenicity for healthy humans, but can cause substantial disease and death in susceptible animals. In line with the clinical symptoms caused in susceptible animals by the two strains and their derived derivatives, the Armstrong strain is classified as neurotropic, in contrast to the WE strain, which is classified as viscerotropic.

[0006] The LCMV genome consists of two negative-stranded RNA segments, designated S (small) and L (large). The S segment encodes the surface glycoprotein (GP) and nucleocapsid protein (NP), while the L segment encodes the viral polymerase (LP) and Z protein (Radoshitzky et al. (2020) in DM Knipe and PM Howley (eds.), Fields Virology (Non-Patent Document 1)).

[0007] Certain LCMV WE strain derivatives have been described as effective agents for solid tumor treatment (WO 2016 / 166285 A1, WO 2020 / 053324 A1). However, the LCMV strain WE has a Biosafety Level (BSL) 3 classification by default, making the manufacture and application of such strains under BSL 3 conditions virtually impossible, thus preventing the use of such products.

[0008] Thus, there is an unmet need to provide viruses with a higher level of attenuation while having comparable or even improved antitumor properties compared to LCMV strain WE.It is therefore an object of the present invention to provide additional and / or improved viruses useful in therapy. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO 2016 / 166285 A1 [Patent Document 2] WO 2020 / 053324 A1 [Non-patent literature]

[0010] [Non-Patent Document 1] Radoshitzky et al. (2020) in DM Knipe and PM Howley (eds.), Fields Virology Summary of the Invention

[0011] The present invention relates to a viral particle comprising a Lymphocytic Choriomeningitis Virus (LCMV) S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40, wherein the glycoprotein comprises at least one mutated amino acid residue compared to the glycoprotein sequence set forth in SEQ ID NO:4.

[0012] The present invention also relates to a viral particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40, wherein the L protein comprises an amino acid residue other than Lys at position 1079 corresponding to the linear polypeptide sequence of SEQ ID NO:40.

[0013] The present invention also relates to a viral particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and an open reading frame encoding a nucleoprotein having at least 97% sequence identity to SEQ ID NO:6, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40, and an open reading frame encoding a Z protein having at least 90% sequence identity to SEQ ID NO:38.

[0014] The present invention also relates to host cells containing an LCMV S segment and an LCMV L segment as contained in the viral particles of the present invention.

[0015] The present invention also relates to a host cell comprising (a) an ORF encoding a glycoprotein, an ORF encoding an L protein, an ORF encoding a nucleoprotein, and an ORF encoding a Z protein, as contained in a viral particle of the present invention; and / or (b) cDNAs of an LCMV S segment and an LCMV L segment, as contained in a viral particle of the present invention.

[0016] The present invention also relates to a method for producing a viral particle of the invention, comprising the step of culturing a host cell of the invention under conditions suitable for viral particle formation.

[0017] The present invention also relates to a pharmaceutical composition comprising a viral particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40.

[0018] The present invention also relates to a viral particle comprising an LCMV S segment and an LCMV L segment, for use in therapy, particularly in the treatment of cancer, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40. [Brief description of the drawings]

[0019] [Figure 1]Mouse B16 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, tumor growth measurements over 7 days and tumor burden on day 7 (n=5 animals / group), one-way ANOVA and Dunnett's multiple comparison test for statistics. [Diagram 2] Murine B16 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, CD8 T cells by flow cytometry from tumors on day 7 (n=5 animals / group). [Diagram 3] Murine B16 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, virus-specific (GP33) CD8 T cells by flow cytometry from tumors on day 7 (n=5 animals / group). [Figure 4] Murine MC38 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, tumor growth measurements over 11 days (n=4 animals / group), t-test for statistics. [Diagram 5] Murine MC38 tumor-bearing Bl / 6 mice were inoculated with LCMV wild-type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, and tumor-infiltrating T cells (CD3+) were measured by flow cytometry on day 11 (n=4 animals / group). [Figure 6] Murine MC38 tumor-bearing Bl / 6 mice were inoculated with LCMV wild-type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, and tumor-infiltrating cytotoxic T cells (CD3+CD8+) were measured by flow cytometry on day 11 (n=4 animals / group). [Figure 7] Murine MC38 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, and tumor immunohistochemistry (IHC) for LCMV (NP protein+) CD8 T cells (CD8+) on day 11 (n=(3)4 animals / group). [Figure 8]In vitro replication of wild-type (WE, clone 13) and recombinant viruses at an MOI=0.1 for 20 h by flow cytometry (NP protein) in HEK293T and human tumor cell lines H1975 (lung), SkMM (myeloma), Ma-Mel-51 (melanoma), C643 (thyroid) and mouse tumor cell lines MC-57 (fibrosarcoma) and B16F10 (melanoma). [Figure 9] In vitro replication of wild-type (WE) and recombinant WE-Cl13 viruses at an MOI of 0.1 for 24 or 48 h in human tumor cell lines H1975 (lung), Mamel 86a and 51 (melanoma), C643 (thyroid), mouse tumor cell lines 511950 (pancreas), Tramp-C2 (prostate), B16.F10 (melanoma), and the mouse dendritic cell line JAWSII by colony formation assay on MC57 cells. [Figure 10] In vitro replication of wild-type (WE) and recombinant viruses at MOI=0.1 for 48 h by replication assays in human primary neurons, mouse tumor cell line MC-57 (fibrosarcoma), mouse MC57G cells, human lung adenocarcinoma cell line A549 and human thyroid carcinoma cell line FTC133. Line: initial inoculation. [Figure 11] Bone marrow-derived dendritic cells. [Figure 12] Treatment of Bl / 6 mice with 2x10E4 PFU / animal iv, serum cytokines on days 1 and 3 pi. The dotted line indicates the lower limit of detection. [Figure 13] Treatment of Bl / 6 mice with 2x10E4 PFU / animal iv, serum cytokines on days 1 and 3 pi. The dotted line indicates the lower limit of detection. [Figure 14] mouse strains with LCMV wild-type (WE, clone 13, Arm) and recombinant (clone 13 L, WE S) strains at 2x10E6 PFU / animal iv on day 0 (n=3 animals / group). [Figure 15] mouse strains with LCMV wild-type (WE, clone 13, Arm) and recombinant (clone 13 L, WE S) strains at 2x10E6 PFU / animal iv on day 0 (n=3 animals / group). [Figure 16] mouse strains with LCMV wild-type (WE, clone 13, Arm) and recombinant (clone 13 L, WE S) strains at 2x10E6 PFU / animal iv on day 0 (n=3 animals / group). [Figure 17] mouse strains with LCMV wild-type (WE, clone 13, Arm) and recombinant (clone 13 L, WE S) strains at 2x10E6 PFU / animal iv on day 0 (n=3 animals / group). [Figure 18] ALT / AST serum levels upon iv treatment of B16 melanoma-bearing Bl / 6 animals with 2x10E4 PFU / animal on day 7 pi. [Figure 19] Murine B16 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, colony formation assays from the indicated organs on day 7 (n=5 animals / group). [Figure 20] Murine MC38 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, and plaque formation assays from the indicated organs on day 11 (n=4 animals / group). [Figure 21] In vitro replication of wild type (WE) and recombinant viruses at MOI=0.1 by flow cytometry (NP protein) in human tumor cell lines H1975 (lung), GIST-T1 (gastrointestinal), Ma-Mel-86a and 51 (melanoma) and mouse tumor cell line MC57G (fibrosarcoma) for 20 h. Line: Replication of LCMV strain WE. [Figure 22] In vitro replication of wild type (WE) and recombinant viruses at MOI=0.1 by flow cytometry (NP protein) in human tumor cell lines H1975 (lung), GIST-T1 (gastrointestinal), Ma-Mel-86a and 51 (melanoma) and mouse tumor cell line MC57G (fibrosarcoma) for 20 h. Line: Replication of LCMV strain WE. [Diagram 23]Differentiated human myoblasts, human skeletal myoblasts (SkMM), lung adenocarcinoma cell line A549 and mouse fibrosarcoma cell line MC57G were seeded (100.000 cells / well) and infected with LCMV strains WE, WE-Cl13(L), ​​P52-Cl13(L), ​​I181M-Cl13(L) or R185W-Cl13(L) at MOI=0.1. After 20 h, cells were stained intracellularly for LCMV nucleoprotein (NP, clone VL4). The percentage of LCMV infected cells (LCMV NP+) was analyzed by flow cytometry. Line: Replication of LCMV strain WE. [Figure 24] In vitro replication of wild type (WE) and recombinant viruses at MOI=0.1 for 48 h by replication assay in human primary neurons and in the mouse tumor cell line MC57G (fibrosarcoma) as a comparison. Line: initial inoculation. [Diagram 25] Mouse B16 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, tumor growth measurements over 7 days and tumor burden on day 7 (n=5 animals / group), one-way ANOVA and Dunnett's multiple comparison test for statistics. [Figure 26] Murine B16 tumor-bearing Bl / 6 mice, LCMV wild-type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, virus-specific (GP33) and CD8 T cells by flow cytometry from dLN and tumors on day 7 (n=5 animals / group). [Figure 27] Murine B16 tumor-bearing Bl / 6 mice, LCMV wild-type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, virus-specific (GP33) and CD8 T cells by flow cytometry from dLN and tumors on day 7 (n=5 animals / group). [Figure 28] Murine B16 tumor-bearing Bl / 6 mice, LCMV wild-type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, virus-specific (GP33) and CD8 T cells by flow cytometry from dLN and tumors on day 7 (n=5 animals / group). [Figure 29]Murine B16 tumor-bearing Bl / 6 mice, LCMV wild-type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, virus-specific (GP33) and CD8 T cells by flow cytometry from dLN and tumors on day 7 (n=5 animals / group). [Diagram 30] Murine MC38 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, tumor growth measurements over 11 days (n=4 animals / group), t-test for statistics. [Diagram 31] Mouse MC38 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, tumor T cell measurement by flow cytometry on day 11, n=4 animals / arm, excluding P52-FP7-Cl13(L) group (n=3) due to experimental error (values ​​near / below control). [Diagram 32] Mouse MC38 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, tumor helper T cell measurement by flow cytometry on day 11, n=4 animals / arm, excluding P52-FP7-Cl13(L) group (n=3) due to experimental error (values ​​near / below control). [Diagram 33] Mouse MC38 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, tumor cytotoxic T cell measurement by flow cytometry on day 11, n=4 animals / arm, excluding P52-FP7-Cl13(L) group (n=3) due to experimental error (values ​​near / below control). [Diagram 34] Naïve Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, cytokine LegendPlex on days 2 and 4 (n=3 animals / group). [Diagram 35] Mouse B16 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, plaque formation assays from the indicated organs on day 7 (n=5 animals / group). [Diagram 36] Murine MC38 tumor-bearing Bl / 6 mice, LCMV wild type (WE) and recombinant strains at 2x10E4 PFU / animal iv on day 0, and plaque formation assays from the indicated organs on day 11 (n=4 animals / group). [Figure 37] FreeStyle 293-F suspension cells were infected with LCMV strains WE-Cl13(L), ​​P52-Cl13(L) or P52-WE(L) at an MOI of 0.001 at a density of 1.5x106 cells per mL. Supernatants were harvested at 12, 24, 30, 34, 40, 48, 72 and 96 hours post-infection. LCMV titers in the supernatants were analyzed by plaque formation assay. [Figure 38-1] FIG. 38: Nucleic acid and amino acid sequences of the L segment, S segment, GPC, NP, ZP, and LP of LCMV strains A. WE, B. P52, C. Armstrong, and D. Clone 13. [Figure 38-2] See description of Figure 38-1. [Figure 38-3] See description of Figure 38-1. [Figure 38-4] See description of Figure 38-1. [Figure 38-5] See description of Figure 38-1. [Figure 38-6] See description of Figure 38-1. [Figure 38-7] See description of Figure 38-1. [Figure 38-8] See description of Figure 38-1. [Figure 38-9] See description of Figure 38-1. [Figure 38-10] See description of Figure 38-1. [Figure 38-11] See description of Figure 38-1. [Figure 38-12] See description of Figure 38-1. [Figure 38-13] See description of Figure 38-1. [Figure 38-14] See description of Figure 38-1. [Figure 38-15] See description of Figure 38-1. [Figure 38-16] See description of Figure 38-1. [Figure 38-17] See description of Figure 38-1. [Figure 38-18] See description of Figure 38-1. [Figure 38-19] See description of Figure 38-1. [Figure 38-20] See description of Figure 38-1. [Figure 38-21] See description of Figure 38-1. [Figure 38-22] See description of Figure 38-1. [Figure 38-23] See description of Figure 38-1. [Figure 38-24] See description of Figure 38-1. [Figure 38-25] See description of Figure 38-1. [Figure 38-26] See description of Figure 38-1. [Figure 38-27] See description of Figure 38-1. [Figure 38-28] See description of Figure 38-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description The inventors of the present application believe that the virulence of the WE strain may be consistently determined by elements located on the L segment, while the S segments of WE and other LCMV strains do not contribute to virulence. Therefore, replacement of the L segment of the WE strain with the corresponding segment of another LCMV strain, such as Armstrong, Clone 13, and their derivatives, would result in a reassorted laboratory strain that no longer exhibits the original virulence profile, including hepatopathogenicity in NHPs and other susceptible animals.

[0021] The inventors of the present application then surprisingly found that a viral strain comprising elements of the S segment of the LCMV WE strain or a derivative thereof that are not believed to contribute to pathogenicity and elements of the L segment of the strains Clone 13 or Armstrong or a derivative thereof that are not believed to contribute to pathogenicity has improved properties compared to the LCMV WE strain, particularly in tumor treatment. Such virus strains may no longer be capable of inducing LCMV-specific disease, including liver disease, in NHPs, rodents or guinea pigs (Riviere et al. (1985) Journal of virology, 55: 704-09; Riviere et al. (1986) Med Microbiol Immunol, 175: 191-2; Baccala et al. (2014) Proc Natl Acad Sci USA, 111: 8925-30; Oldstone et al. (2018) Proceedings of the National Academy of Sciences, 115: E7814).

[0022] Studies carried out by the inventors of the present application using naive and susceptible rodents indeed demonstrate that the reassortant viruses according to the present invention may have reduced pathogenic properties compared to the LCMV WE strain, including hepatopathogenicity (Examples 16, 17, 18, 19, and 35). These results confirm that the viral particles of the present invention may result in virus-based products that can be safely handled during manufacturing and clinical trials, due to the reduced pathogenic properties described for wild-type LCMV strains, including hepatopathogenesis and neurotropism (Examples 10, 21, 24).

[0023] Under its significantly reduced replicative potential (up to >100-fold) in cells of different origins (Examples 8 and 9), the viral particles of the present invention can show a significantly reduced potential to induce in vivo pathogenic cytokines in naive animals, while lacking any signs of induced organ pathogenicity (Examples 12-17). In particular, only the viral particles containing the WE strain-derived S segment element and the Clone 13 strain-derived L segment element showed attenuated replication in Example 8, but the viral particles containing the Clone 13 strain-derived S segment element and the WE strain-derived L segment element did not, which supports the hypothesis that the pathogenicity of the WE and Clone 13 strains is determined by their L or S segments, respectively.

[0024] The FVB mouse strain, which shows a predisposition to virus-induced pathology (Schnell et al. (2012) PLoS Pathog, 8: e1003073), succumbs to a hemorrhagic fever-like disease including thrombocytopenia and hepatocyte necrosis when infected with LCMV. Importantly, the disease in FVB mice mimics the clinical signs of LCMV disease and Argentine hemorrhagic fever in macaques (Schnell et al. (2012) PLoS Pathog, 8: e1003073). However, in contrast to LCMV strains WE, clone 13, and Armstrong, infection with the virus particles of the present invention did not result in loss of animal weight or any other signs of liver pathology or hemorrhagic fever-like disease in Examples 14-17.

[0025] An important and surprising feature of the viral particles of the invention for clinical development is that, despite their attenuation, they retain or even enhance their strong antitumor effect, as described for the WE strain (Kalkavan et al. (2017) Nat Commun, 8: 14447). The viral particles of the invention may be equipped with further modifications to increase their tumor tropism (Examples 21-23). ​​Such modifications may include mutations at positions 181 and / or 185 of the GP, in particular Arg 185→Trp and / or Ile 181→Met. Indeed, such viral particles may show strong antitumor effects in mouse tumor models (Example 25), but have the attenuated phenotype of the WE-clone 13 strain from which they are derived (Examples 34 and 38). The pathogenicity of LCMV is mainly dictated by the antiviral function of the immune system and has been reported to correlate with the intensity of viral replication in infected organs (Lang et al. (2010) Cell Physiol Biochem, 26: 263-72). Compared with the WE strain, such modified viral particles may result in a stronger early LCMV-directed T cell response, the main antiviral immune effector mechanism, and its organ distribution is already controlled early in infection (Example 35).

[0026] In previous publications, it was described that LCMV can induce organ damage (e.g., liver) due to excessive and deregulated innate (Th1 and inflammatory cytokines) and adaptive (CD8 T cell) immune responses (Lang et al. (2010) Cell Physiol Biochem, 26: 263-72; Schnell et al. (2012) PLoS Pathog, 8: e1003073; Oldstone et al. (2018) Proceedings of the National Academy of Sciences, 115: E7814). The inventors of the present application surprisingly found that, although a similar cytokine response pattern was observed with wild-type LCMV strains Armstrong, Clone 13 and WE, the strength of the immune response for the viral particles of the present invention may be substantially lower. This observation is particularly important for IFN-α, where high cytokine levels early in infection were associated with organ damage in the respective animal models (Schnell et al. (2012) PLoS Pathog, 8: e1003073). The viral particles of the present invention, in contrast, can induce substantially lower IFN-α and IFN-γ levels, especially early in infection (Examples 12 and 13), strongly pointing to a significantly lower potential for IFN-mediated adverse effects (Baccala et al. (2014) Proc Natl Acad Sci USA, 111: 8925-30). In addition, the inflammatory cytokines IL-6 and TNF-α, as well as IL-10, could also be substantially decreased at different time points (days 1 and 3) during infection, indicating enhanced viral control (Lang et al. (2010) Cell Physiol Biochem, 26: 263-72; Schnell et al. (2012) PLoS Pathog, 8: e1003073).

[0027] Overall, the viral particles of the present invention may retain or even enhance their anti-tumor efficacy, yet exhibit a significantly attenuated phenotype, and even a more potent in vivo attenuated immune phenotype.

[0028] Thus, the present invention relates to a viral particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40.

[0029] The S segment can also contain an open reading frame encoding a nuclear protein. The nuclear protein can have at least 97% sequence identity to SEQ ID NO:6.

[0030] The L segment can also include an L segment. The L segment can include an open reading frame encoding a Z protein having at least 90% sequence identity to SEQ ID NO:38.

[0031] The viral particles of the present invention are preferably arenavirus particles, more preferably lymphocytic choriomeningitis virus (LCMV) particles.

[0032] Wild-type arenavirus genome segments and ORFs are known in the art. In particular, arenavirus genome consists of S segment and L segment. S segment carries ORFs that code for GP and NP. L segment codes for L protein and Z protein. Both segments are flanked by their respective 5' and 3' UTRs.

[0033] The viral particles of the present invention preferably comprise genome segments corresponding to those of wild-type arenaviruses. This means that the S segment carries the ORFs encoding GP and NP, and the L segment encodes the L and Z proteins. In the S segment, the ORF encoding the glycoprotein is under the control of the 5' untranslated region (UTR), and the nucleoprotein is under the control of the 3' UTR. In the L segment, the ORF encoding the L protein is under the control of the 3' UTR, and the ORF encoding the Z protein is under the control of the 5' UTR. Thus, the genes of the viral particles of the present disclosure are preferably located in their natural locations. Thus, the viral particles of the present invention preferably have a bipartite genome. The genome of the viral particles of the present invention preferably consists of one L segment and one S segment as described herein. Exemplary examples of LCMV S segments are shown in SEQ ID NOs: 1, 11, 21, and 31. Exemplary examples of LCMV L segments are shown in SEQ ID NOs: 2, 12, 22, and 32.

[0034] Several strains of LCMV are part of this disclosure. As used herein, LCMV "WE", "strain WE", "WE strain", etc. refer to LCMV having genome segments as shown in SEQ ID NOs: 1 and 2. As used herein, LCMV "P52", "P52-WE", "strain P52", "strain P52", etc. refer to mutants / derivatives of the WE strain having genome segments as shown in SEQ ID NOs: 11 and 12. As used herein, LCMV "Armstrong", "strain Armstrong", "Armstrong strain", etc. refer to LCMV strain Armstrong 53b having genome segments as shown in SEQ ID NOs: 21 and 22. As used herein, LCMV "clone 13", "Cl13", "strain clone 13", "strain clone 13", etc. refer to LCMV having genome segments as shown in SEQ ID NOs: 31 and 32.

[0035] The terms "glycoprotein," "GP," and "G protein" are used interchangeably and refer to LCMV-derived glycoproteins that are believed to mediate receptor binding and membrane fusion. Exemplary examples of glycoproteins are set forth in SEQ ID NOs:4, 14, 24, and 34. Exemplary examples of genes encoding glycoproteins are set forth in SEQ ID NOs:3, 13, 23, and 33.

[0036] The terms "L protein" and "LP" are used interchangeably and refer to the RNA polymerase L from LCMV. Exemplary examples of L proteins are set forth in SEQ ID NOs:10, 20, 30, and 40. Exemplary examples of genes encoding L proteins are set forth in SEQ ID NOs:9, 19, 29, and 39.

[0037] The terms "nucleoprotein," "N protein," and "NP" are used interchangeably and refer to LCMV-derived nucleoprotein. Exemplary examples of nucleoproteins are set forth in SEQ ID NOs:6, 16, 26, and 36. Exemplary examples of genes encoding nucleoproteins are set forth in SEQ ID NOs:5, 15, 25, and 35.

[0038] The terms "Z protein" or "ZP" are used interchangeably and refer to the LCMV-derived small RING finger protein Z. Exemplary examples of Z proteins are shown in SEQ ID NOs:8, 18, 28, and 38. Exemplary examples of genes encoding Z proteins are shown in SEQ ID NOs:7, 17, 27, and 37.

[0039] The viral particles of the invention comprise a glycoprotein, wherein the glycoprotein may comprise at least one mutated amino acid residue compared to the glycoprotein sequence set forth in SEQ ID NO:4. For example, the glycoprotein may comprise at least one mutated amino acid residue at positions 181 and / or 185 compared to the glycoprotein sequence set forth in SEQ ID NO:4. However, it is preferred that the glycoprotein comprises mutations at both positions compared to SEQ ID NO:4. Preferred mutations at these positions are selected from Arg 185→Trp and Ile 181→Met. Thus, the viral particles may have either or, preferably, both mutations.

[0040] Without wishing to be bound by theory, it is believed that the presence of one or two of the above-mentioned mutations in the glycoprotein can improve the function (e.g., anti-tumor activity) of LCMV, regardless of the presence of other mutations, particularly other mutations in other genes or proteins of LCMV.

[0041] Viral particles of the invention comprise an L protein, wherein the L protein may comprise an amino acid residue other than Lys at position 1079, which corresponds to the linear polypeptide sequence of SEQ ID NO: 40. The amino acid residue at this position is preferably a non-basic amino acid residue, more preferably a neutral hydrophilic amino acid residue, even more preferably Asn or Gln, and most preferably Gln.

[0042] The term "amino acid" or "amino acid residue" refers to an amino acid having its art-recognized definition, such as an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used if desired. Naturally occurring residues are divided into groups based on common side chain properties: (1) hydrophobic: methionine, alanine, valine, leucine, isoleucine; (2) neutral hydrophilic: cysteine, serine, threonine, asparagine, glutamine; (3) acidic: aspartic acid, glutamic acid; (4) basic: histidine, lysine, arginine; (5) residues that affect chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine. In some embodiments, substitutions may involve exchanging a member of one of these classes for another.

[0043] The viral particles of the present invention comprise an S segment and an L segment, wherein the S segment is preferably derived from LCMV strain WE or a mutant thereof, and wherein the L segment is preferably derived from LCMV strain clone 13 or a mutant thereof.

[0044] The L segment variant may have at least about 83%, preferably at least about 84%, preferably at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity to the sequence of the original L segment.

[0045] The viral particles disclosed herein may have an identity of at least about 83%, preferably at least about 84%, preferably at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% identical to the sequence set forth in SEQ ID NO:21 or 31. Preferably, the L segment comprises or preferably consists of a sequence having or preferably being identical to at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity.

[0046] A variant of the S segment may have at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity to the sequence of the original S segment.

[0047] The viral particles disclosed herein may have an S segment comprising, or preferably consisting of, a sequence having or preferably identical to a sequence set forth in SEQ ID NO:1 or 11, at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity to or preferably identical to a sequence set forth in SEQ ID NO:1 or 11.

[0048] Viral particles of the invention comprising LCMV preferably comprise an S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and an open reading frame encoding a nucleoprotein having at least 97% sequence identity to SEQ ID NO:6, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40, and an open reading frame encoding a Z protein having at least 90% sequence identity to SEQ ID NO:38.

[0049] An open reading frame encoding a glycoprotein disclosed herein may encode a sequence that has at least about 98%, preferably at least about 98.5%, preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.5%, preferably at least about 99.7% sequence identity to or preferably identical to a sequence set forth in SEQ ID NO:4 or 14.

[0050] An open reading frame encoding a glycoprotein disclosed herein may comprise a sequence having or preferably being identical to a sequence set forth in SEQ ID NO:3 or 13, with at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity.

[0051] The open reading frame encoding the L protein disclosed herein may encode a sequence that has or is preferably identical to the sequence set forth in SEQ ID NO:30 or 40, with at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98%, preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity.

[0052] The open reading frame encoding the L protein disclosed herein may have or is preferably identical to the sequence set forth in SEQ ID NO:29 or 39, with at least about 83%, preferably at least about 84%, preferably at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity.

[0053] An open reading frame encoding a nuclear protein disclosed herein may encode a sequence having at least about 98%, preferably at least about 98.5%, preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity to or preferably identical to a sequence set forth in either SEQ ID NO:6 or 16.

[0054] An open reading frame encoding a nuclear protein disclosed herein may have, or is preferably identical to, a sequence set forth in SEQ ID NO:5 or 15, of at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity.

[0055] The open reading frame encoding the Z protein disclosed herein may encode a sequence having or preferably being identical to the sequence set forth in SEQ ID NO:28 or 38, which has at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98%, preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity.

[0056] The open reading frame encoding the Z protein disclosed herein may have or be identical to the sequence set forth in SEQ ID NO: 27 or 37, with at least about 84%, preferably at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity.

[0057] Preferred viral particles comprise an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 98.5% sequence identity to SEQ ID NO:4 or 14 (SEQ ID NO:14 is preferred), and an open reading frame encoding a nucleoprotein having at least 98.5% sequence identity to SEQ ID NO:6 or 16 (SEQ ID NO:16 is preferred), and wherein the L segment comprises an open reading frame encoding an L protein having at least 95% sequence identity to SEQ ID NO:30 or 40 (SEQ ID NO:40 is preferred), and an open reading frame encoding a Z protein having at least 95% sequence identity to SEQ ID NO:28 or 38 (SEQ ID NO:38 is preferred).

[0058] Preferred viral particles comprise an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 98% sequence identity to SEQ ID NO:4 or 14 (SEQ ID NO:14 is preferred), and an open reading frame encoding a nucleoprotein having at least 98% sequence identity to SEQ ID NO:6 or 16 (SEQ ID NO:16 is preferred), and wherein the L segment comprises an open reading frame encoding an L protein having at least 98% sequence identity to SEQ ID NO:30 or 40 (SEQ ID NO:40 is preferred), and an open reading frame encoding a Z protein having at least 98% sequence identity to SEQ ID NO:28 or 38 (SEQ ID NO:38 is preferred).

[0059] Preferred viral particles comprise an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 99% sequence identity to SEQ ID NO:4 or 14 (SEQ ID NO:14 is preferred), and an open reading frame encoding a nucleoprotein having at least 99% sequence identity to SEQ ID NO:6 or 16 (SEQ ID NO:16 is preferred), and wherein the L segment comprises an open reading frame encoding an L protein having at least 99% sequence identity to SEQ ID NO:30 or 40 (SEQ ID NO:40 is preferred), and an open reading frame encoding a Z protein having at least 99% sequence identity to SEQ ID NO:28 or 38 (SEQ ID NO:38 is preferred).

[0060] Preferred viral particles comprise an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having the sequence set forth in SEQ ID NO:4 or 14 (SEQ ID NO:14 is preferred), and an open reading frame encoding a nucleoprotein having the sequence set forth in SEQ ID NO:6 or 16 (SEQ ID NO:16 is preferred), and wherein the L segment comprises an open reading frame encoding an L protein having the sequence set forth in SEQ ID NO:30 or 40 (SEQ ID NO:40 is preferred), and an open reading frame encoding a Z protein having the sequence set forth in SEQ ID NO:28 or 38 (SEQ ID NO:38 is preferred).

[0061] A preferred viral particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment contains an open reading frame encoding a glycoprotein having the sequence set forth in SEQ ID NO:4, and an open reading frame encoding a nucleoprotein having the sequence set forth in SEQ ID NO:6, and wherein the L segment contains an open reading frame encoding an L protein having the sequence set forth in SEQ ID NO:40, and an open reading frame encoding a Z protein having the sequence set forth in SEQ ID NO:38.

[0062] A preferred viral particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment contains an open reading frame encoding a glycoprotein having the sequence set forth in SEQ ID NO:14, and an open reading frame encoding a nucleoprotein having the sequence set forth in SEQ ID NO:16, and wherein the L segment contains an open reading frame encoding an L protein having the sequence set forth in SEQ ID NO:40, and an open reading frame encoding a Z protein having the sequence set forth in SEQ ID NO:38.

[0063] A preferred viral particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment contains an open reading frame encoding a glycoprotein having the sequence set forth in SEQ ID NO:4, and an open reading frame encoding a nucleoprotein having the sequence set forth in SEQ ID NO:6, and wherein the L segment contains an open reading frame encoding an L protein having the sequence set forth in SEQ ID NO:30, and an open reading frame encoding a Z protein having the sequence set forth in SEQ ID NO:28.

[0064] A preferred viral particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment contains an open reading frame encoding a glycoprotein having the sequence set forth in SEQ ID NO:14, and an open reading frame encoding a nucleoprotein having the sequence set forth in SEQ ID NO:16, and wherein the L segment contains an open reading frame encoding an L protein having the sequence set forth in SEQ ID NO:30, and an open reading frame encoding a Z protein having the sequence set forth in SEQ ID NO:28.

[0065] The viral particles of the present disclosure are preferably free of heterologous ORFs. "Heterologous ORFs" in this context refers to ORFs derived from organisms other than LCMV and / or ORFs encoding artificial or synthetic proteins.

[0066] The term "nucleic acid" as used herein may broadly refer to DNA or RNA. DNA and RNA differ, among other things, in their nucleobases. In DNA, the complementary base of adenine is thymine, whereas in RNA, it is uracil. For simplicity, throughout this specification, the base corresponding to adenine is designated as "t", which can refer to thymine (in DNA) or uracil (in RNA) depending on the context.

[0067] The terms "encoding" or "encoding," as used herein in the context of nucleic acids, refer to a nucleic acid having a sequence that can be translated into a specific amino acid sequence encoded by the nucleic acid. A nucleic acid sequence can include the sequence of the coding strand and can also include the sequence of the strand complementary to the coding strand.

[0068] "Percent sequence identity" with respect to the sequences disclosed herein is defined as the percentage of amino acid residues or nucleotides of a candidate sequence that are pairwise identical to those of a reference sequence, after alignment and introduction of gaps as necessary to obtain the maximum percent sequence identity, and without considering conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved by any method in the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. This also applies to the nucleotide sequences disclosed herein. For purposes of determining sequence identity, uracil (e.g., in the case of RNA) may be considered equivalent to thymine (e.g., in the case of DNA).

[0069] The term "attenuated" as used herein relates to a reduced ability of a virus to replicate in a given host (cell), to replicate in healthy organs, and / or to induce cytokines. The viral particles of the present invention are preferably attenuated.

[0070] The term "infectious" as used herein relates to the ability of a virus to infect, i.e. to enter, a given host cell. The viral particles of the present invention are preferably infectious.

[0071] The term "replication competent" means that the virus has the ability to amplify and express its genetic material in infected cells and can produce further progeny in normal cells that are not genetically engineered. In particular, a replication competent virus does not require a host cell that is engineered to express viral genes in order to be able to replicate. The viral particles of the present invention are preferably replication competent.

[0072] The term "pathogenic" as used herein refers to the ability of a virus to cause disease, i.e., to cause harm to an organism.

[0073] The viral particles of the invention may be capable of promoting at least the same or even greater reduction in tumor growth as compared to LCMV strain WE and / or viral particles comprising the same S segment as said viral particles and the L segment as shown in SEQ ID NO: 2. In particular, the viral particles may be capable of promoting a greater reduction in cold tumor growth as compared to LCMV strain WE and / or viral particles comprising the same S segment as said viral particles and the L segment as shown in SEQ ID NO: 2, preferably as measured in an assay essentially as described in Example 1.

[0074] The viral particles of the present invention may be capable of inducing adaptive immune activation that is at least as strong or even stronger than that of LCMV strain WE and / or viral particles comprising the same S segment as said viral particles and the L segment shown in SEQ ID NO:2, preferably as measured in an assay essentially as described in Example 3.

[0075] The viral particles of the present invention may be capable of promoting at least the same degree of reduction in the growth of warm tumors as compared to LCMV strain WE and / or viral particles comprising the same S segment as said viral particles and the L segment shown in SEQ ID NO:2, preferably as measured in an assay essentially as described in Example 4.

[0076] As used herein, "hot" or "warm" tumors are used interchangeably and refer to tumors with T cell inflammatory phenotype.Such tumors show signs of inflammation, which means that the tumor is already infiltrated by T cells to fight cancer cells.Examples of cancers that are typically hot tumors include melanoma, bladder cancer, kidney cancer, head and neck cancer, and non-small cell lung cancer.

[0077] As used herein, "cold tumor" refers to a tumor with a non-T cell inflammatory phenotype, with low or no T cell infiltration. Due to the lack of T cells, it is difficult to induce an immune response with immunotherapeutic drugs in such tumors. Examples of cancers that are typically cold tumors include breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and glioblastoma. The difference between hot and cold tumors is described in detail in Gajewski et al. (2017) Adv Exp Med Biol. 1036:19-31 and Maleki Vareki (2018) Journal for ImmunoTherapy of Cancer 6:157.

[0078] The viral particles of the invention may be capable of inducing reduced levels of interferon upon (preferably systemic) administration, preferably as measured in an assay essentially as described in Example 12, compared to LCMV strains WE, Armstrong, Clone 13, and / or viral particles comprising the same S segment as said viral particles and the L segment shown in SEQ ID NO:2. The interferon is preferably selected from the group consisting of IFN-α and IFN-β.

[0079] The viral particles of the invention may be capable of inducing lower concentrations of one or more Th1 and inflammatory cytokines as compared to LCMV strains WE, Armstrong, Clone 13, and / or viral particles comprising the same S segment as said viral particles and the L segment shown in SEQ ID NO:2, preferably as measured in an assay essentially as described in Example 13. The cytokines are preferably selected from the group consisting of IL-6, IL-10, TNFα, and IFNγ.

[0080] The viral particles of the invention may be capable of inducing lower concentrations of one or more Th1 and inflammatory cytokines as compared to LCMV strains WE, Armstrong, Clone 13, and / or viral particles comprising the same S segment as said viral particles and the L segment shown in SEQ ID NO:2, preferably as measured in an assay essentially as described in Example 13. The cytokines are preferably selected from the group consisting of IL-6, IL-10, TNFα, and IFNγ.

[0081] Viral particles of the invention may preferably be less pathogenic in mice compared to LCMV strain WE, as measured in FVB / N mice in an assay essentially as described in Example 14.

[0082] The viral particles of the present invention may be capable of inducing a reduced degree of liver pathology in mice, preferably as measured in an assay essentially as described in Example 18, compared to LCMV strain WE and / or viral particles comprising the same S segment as said viral particles and the L segment shown in SEQ ID NO:2.

[0083] The viral particles of the present invention may be capable of inducing a reduced degree of thrombocytopenia in mice, or may be capable of inducing undetectable thrombocytopenia in mice (e.g., FVB / N mice), as compared to LCMV strain WE and / or viral particles comprising the same S segment as said viral particles and the L segment shown in SEQ ID NO:2, preferably as measured in an assay essentially as described in Example 15.

[0084] The viral particles of the present invention may exhibit reduced replication in healthy cells (e.g., neuronal cells) compared to LCMV strain WE and / or viral particles comprising the same S segment as said viral particles and the L segment shown in SEQ ID NO:2, preferably as measured in an assay essentially as described in Example 24.

[0085] Viral particles of the invention may preferably exhibit increased replication in tumor cells compared to LCMV strain WE, as measured in an assay essentially as described in Example 24.

[0086] The viral particles of the present invention may exhibit reduced replication in healthy organs, preferably as measured in an assay essentially as described in Example 35, compared to LCMV strain WE and / or viral particles comprising the same S segment as said viral particles and the L segment shown in SEQ ID NO:2.

[0087] The present invention also relates to host cells that contain an LCMV S segment and an LCMV L segment as described for the viral particles of the present invention.

[0088] The present invention also relates to a host cell comprising cDNA of an ORF encoding a glycoprotein as described for the viral particle of the present invention, an ORF encoding an L protein as described for the viral particle of the present invention, an ORF encoding a nucleoprotein as described for the viral particle of the present invention, and an ORF encoding a Z protein as described for the viral particle of the present invention.

[0089] The present invention also relates to host cells which contain the LCMV S segment and LCMV L segment cDNAs as described for the viral particles of the invention.

[0090] The techniques for generating cDNA are routine and conventional techniques of molecular biology and DNA manipulation and generation. Any cloning technique known to those skilled in the art can be used. Such techniques are well known and available to those skilled in the art in laboratory manuals such as Sambrook and Russell, Molecular Cloning: A laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory NY (2001).

[0091] The cDNA described herein can be incorporated into a plasmid. The cDNA described herein can be part of or incorporated into a DNA expression vector, and can be optionally introduced into a host cell. The cDNA described herein or the plasmid or vector containing the cDNA preferably includes a promoter. Specific examples of promoters include RNA polymerase I promoter, RNA polymerase II promoter, RNA polymerase III promoter, T7 promoter, SP6 promoter, or T3 promoter.

[0092] The host cell can be any host cell suitable for cloning, expression, propagation, or production of viral particles. The host cell can be a prokaryote, such as Escherichia coli (E. coli) or Bacillus subtilis, or a eukaryote, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, an immortalized mammalian cell line (e.g., HEK cells, BHK cells, HeLa cells, or CHO cells), or a primary mammalian cell. Preferred host cells include HEK cells, particularly HEK293 cells, such as HEK293T (CVCL_0063) or FreeStyle 293-F (CVCL_D603). FreeStyle 293-F cells are commercially available, for example, from Thermo Fisher Scientific Inc. (catalog number 12338026). Preferred host cells also include BHK cells, such as BHK-21 cells (CVCL_1914). Preferred host cells also include WHO Vero RCB 10-87 cells (ATCC CCL 81).

[0093] The present invention also relates to a method for producing the viral particles of the invention, the method comprising the step of culturing a host cell of the invention under conditions suitable for viral particle formation.

[0094] The method for producing viral particles may further include the step of introducing the cDNA described herein into a host cell. The method for producing viral particles may also include the recovery and / or purification of viral particles. Such recovery and / or purification methods are well known to those skilled in the art.

[0095] The present invention also relates to a pharmaceutical composition comprising the viral particle disclosed herein. The viral particle preferably comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity with SEQ ID NO:4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity with SEQ ID NO:40. The pharmaceutical composition may further comprise a pharma- ceutically acceptable excipient or carrier. The carrier may be selected from the group consisting of water, saline, buffered aqueous solution, cell culture medium, and a combination of at least two of the aforementioned carriers.

[0096] The present invention also relates to the viral particles disclosed herein for use in therapy. The viral particles preferably comprise an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40. The viral particles disclosed herein can be for use in the treatment of cancer or tumors.

[0097] The present invention also relates to the use of the disclosed viral particles for the manufacture of a medicament. The viral particle preferably comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40. The medicament is preferably for the treatment of cancer or tumor.

[0098] The present invention also relates to a method of treating a disease comprising administering the viral particles disclosed herein to a subject, the subject being preferably in need thereof. The viral particles are preferably administered in an effective amount. The viral particles preferably comprise an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40. The disease is preferably a cancer or a tumor.

[0099] A "subject" is a vertebrate, preferably a mammal, more preferably a human. The term "mammal" is used herein to refer to any animal classified as a mammal, including, but not limited to, humans, domestic and farm animals, as well as zoo, sports, or pet animals, such as mice, sheep, dogs, horses, cats, cows, rats, pigs, monkeys such as cynomolgus monkeys, and the like, to name just a few illustrative examples. Preferably, the mammal herein is a human. The cancer or tumor may be a human or mouse cancer or tumor, with a human cancer or tumor being preferred.

[0100] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations.

[0101] The cancer or tumor may be any cancer or tumor disclosed herein. Generally, the cancer or tumor may be selected from the group consisting of carcinoma, melanoma, blastoma, lymphoma, and sarcoma.

[0102] The term "carcinoma" in the context of the present disclosure should be understood to mean a malignant neoplasm of epithelial origin. The carcinoma is preferably selected from the group consisting of anal cancer, bronchial cancer, lung cancer, endometrial cancer, gallbladder cancer, bladder cancer, hepatocellular carcinoma, testicular cancer, colon cancer, colorectal cancer, rectal cancer, laryngeal cancer, esophageal cancer, gastric cancer, breast cancer, renal cancer, ovarian cancer, pancreatic cancer, pharyngeal cancer, oropharynx cancer, prostate cancer, thyroid cancer and cervical cancer.

[0103] The term "sarcoma" in the context of the present disclosure should be understood to mean a malignant neoplasm of mesodermal origin. The sarcoma may be selected from the group consisting of angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, Kaposi's sarcoma, liposarcoma, leiomyosarcoma, malignant fibrous histiocytoma, neurogenic sarcoma, osteosarcoma and rhabdomyosarcoma.

[0104] The term "melanoma" in the context of the present disclosure should be understood to mean a malignant neoplasm of melanocytic origin.

[0105] The term "lymphoma" in the context of the present disclosure should be understood to mean a malignant neoplasm of lymphocyte origin.

[0106] The term "blastoma" in the context of the present disclosure should be understood to mean a malignant neoplasm of embryonic origin.

[0107] The cancer or tumor to be treated may be a cold tumor. Preferred cold tumors include breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and glioblastoma.

[0108] The cancer or tumor to be treated can be a hot tumor. Preferred hot tumors include melanoma, bladder cancer, kidney cancer, head and neck cancer, and non-small cell lung cancer.

[0109] Preferred cancers or tumors to be treated include melanoma, colon cancer, fibrosarcoma, pancreatic cancer, thyroid cancer, lung cancer, adenocarcinoma, and gastrointestinal cancer.

[0110] Generally, the viral particles disclosed herein can be administered via any suitable route known to those skilled in the art. Administration generally includes enteral and parenteral administration. Parenteral administration can include local administration, such as intramuscular, intraperitoneal, subcutaneous, or intratumoral administration. Alternatively, parenteral administration can include systemic administration, particularly intravenous administration, such as by injection or infusion.

[0111] Unless otherwise indicated, the term "at least" preceding a series of elements is understood to refer to every single element of the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0112] The term "and / or" whenever used herein includes the meaning of "and", "or" and "all or any other combination of the elements with which it connects".

[0113] The terms "about" or "approximately" as used herein mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range, but also include specific numbers, for example, about 20 includes 20.

[0114] Throughout this specification and claims, unless the context specifically contradicts, the word "comprise" and variations such as "comprises" and "comprising" are understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or groups of integers or steps. As used herein, the term "comprising" can be interchanged with the terms "containing" or "including," or, as sometimes used herein, with the term "having."

[0115] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0116] In each instance herein, the terms "comprising," "consisting essentially of," and "consisting of" may be interchanged with either of the other two terms. For example, the term "comprising" also provides explicit support for "consisting essentially of" and "consisting of," the term "consisting essentially of" also provides explicit support for "comprising" and "consisting of," and the term "consisting of" also provides explicit support for "consisting essentially of" and "comprising." EXAMPLES

[0117] mouse Analysis of antitumor effects was performed by using wild-type animals on a C57BL / 6 background. For in vivo analysis of LCMV-induced effects, wild-type mouse strains C57BL / 6, BALB / c or FVB / N were used. C57BL / 6 and BALB / c strains are resistant to lethal infection caused by LCMV. FVB / N mice represent a highly susceptible mouse strain that develops a hemorrhagic fever-like disease after infection with LCMV strain clone 13. After LCMV treatment, habitus was observed and body weight was measured.

[0118] cell line MC57G (CVCL_4985) is a mouse fibrosarcoma cell line that exhibits robust LCMV replication. Tramp-C2 is a mouse prostate cell line (CVCL_3615). B16 (CVCL_F936) and B16-F10 (B16F10, CVCL_0159) are mouse melanoma cell lines. B16-Ova represents the respective B16 cell line expressing ovalbumin as an antigen. MC-38 (MC38, CVCL_B288) is a mouse colon adenocarcinoma cell line. JAWSII is a mouse spontaneously immortalized cell line that can be differentiated into dendritic-like cells by treatment with GM-CSF (CVCL_3727). 511950 is a genetically engineered mouse primary pancreatic cancer cell line with less than 20 passages (Mazur et al. (2015) Nature medicine 21(10): 1163-1171). Bone marrow-derived dendritic cells (BMDCs) were differentiated from mouse bone marrow in the presence of GM-CSF.

[0119] C643 (CVCL_5969) is a human anaplastic thyroid carcinoma cell line. NCI-H1975 (H1975, CVCL_1511) is a human lung adenocarcinoma cell line. A549 is a human adenocarcinoma cell line (CVCL_0023). FTC133 is a human thyroid carcinoma cell line (CVCL_1219). GIST-T1 is a human gastrointestinal tumor cell line derived from pleural effusion (CVCL_4976). Hek293T (CVCL_0063) is a human embryonic kidney cell line that exhibits robust LCMV replication. FreeStyle 293-F (CVCL_D603) is a derivative of Hek293 cells grown in Freestyle medium. FreeStyle 293-F cells are the preferred producer cell line for LCMV production.

[0120] UKE-Ma-Mel-51 (Mamel51, CVCL_A186) and UKE-Ma-Mel-86a (Ma-Mel-86a, CVCL_A221) are human primary melanoma cell lines derived from lymph node metastases provided by Prof. Dr. Paschen, Klinik fur Dermatologie, UK Essen. Primary human neurons were differentiated from human neural progenitor cells (NPCs) kindly provided by Prof. Dr. Gopalakrishnan, HHU Dusseldorf. SkMM (human skeletal muscle myoblasts, Lonza CC-2580) are human primary skeletal myoblasts. Myotubes were differentiated from SkMM in the presence of SkGM-2, horse serum, glutamine, gentamicin and dexamethasone. Differentiation was verified by staining with anti-myosin 4 AK (eBioscience, Clone MF-20).

[0121] BHK-21 cells represent a spontaneously immortalized fibroblast hamster cell line (CVCL_1914).

[0122] virus LCMV strain WE was obtained from the laboratory of Prof. Zinkernagel (Experimental Immunology, Zurich, Switzerland) and has been propagated since 2008 in L929 or BHK-21 cells.

[0123] LCMV strains Clone 13 and Armstrong 53b were obtained from S. Basta, Queens University, and RM Zinkernagel, University of Zurich, respectively. LCMV reassortant WE-Cl13(L), ​​P52-Cl13(L), ​​P52-WE(L), and Cl13-WE(L) were rescued by transient transfection entirely from plasmids as described (Flatz et al. (2006) Proc Natl Acad Sci USA 103(12): 4663-4668). Viruses consist of the S segment of LCMV strain WE or WE-derived P52 and the L segment of LCMV strain Clone 13 or WE. Viruses were propagated in BHK-21 or Freestyle 293F cells.

[0124] Production of recombinant viruses LCMV recombinant and reassortant viruses were generated entirely from plasmids. BHK-21 cells were transiently transfected with plasmids encoding LCMV S-segment, L-segment, and helper plasmids encoding L polymerase and nucleoprotein. LCMV reassortant WE-Cl13(L) was generated using WE S-segment and clone 13 L-segment plasmids. LCMV P52-Cl13(L) was rescued using LCMV P52 S-segment plasmid with two coding mutations in the glycoprotein, I181M and R185W, and clone 13 L-segment plasmid. LCMV P52-WE(L) was generated using LCMV P52 S-segment plasmid and LCMV WE L-segment plasmid. Cl13-WE(L) was generated using LCMV clone 13 S-segment plasmid and WE L-segment plasmid.

[0125] Determination of LCMV-infected cells and immune cell infiltration in ex vivo tumor tissues by immunofluorescence Immunohistofluorescence was used to detect LCMV and immune cell distribution in tumor tissues of LCMV-treated or control-treated tumor-bearing mice.Tumor biopsies were cut into 7 μm-thick sections, stained with fluorochrome-labeled anti-LCMV-NP antibody (clone VL4) and antibody against CD8 (eBioscience), visualized under a fluorescence microscope (Keyence) and photographed with an integrated CCD camera.

[0126] Determination of LCMV in supernatants or organs by plaque formation assay To analyze LCMV replication capacity, tumor and primary cells were seeded in 24-well plates (100.000 cells / well) and infected with LCMV at the indicated MOI. Supernatants were harvested 24, 48 or 72 hours post-infection. Analysis of LCMV production by FreeStyle 293-F cells was performed using 1.5x10 cells per mL. 6 Cells were seeded and infected with LCMV at an MOI of 0.001. Supernatants were harvested 12, 24, 30, 34, 40, 48, 72 and 96 hours after infection. Analysis of LCMV biodistribution was performed in infected tumor-bearing and non-tumor-bearing animals. Organs of infected mice were dissected and homogenized in cell culture medium. Supernatants were titrated and incubated on MC57G cells (100.000 cells / well). A methylcellulose overlay was added after 3-4 h. LCMV-infected plaques were stained with anti-LCMV-NP antibody (clone VL4). Plaques were counted and infectious particles were determined as viral titers per mL of supernatant or per organ.

[0127] Determination of LCMV-infected cells by flow cytometry To analyze LCMV infectivity in tumor and primary cells, cells were seeded in 96-well plates and infected with LCMV at MOI=0.1 or left untreated and incubated on ice for 1 h. After 1 h, cells were transferred to 37°C. 20 h post-infection, cells were harvested and intracellular LCMV staining was performed using fluorescently labeled LCMV NP antibody (clone VL4). Cells were analyzed by flow cytometry (LSR Fortessa, BD) and results were expressed as percent LCMV infected cells or mean fluorescence intensity (MFI).

[0128] Determination of cytokine production by flow cytometry The intensity of immune activation by LCMV was analyzed by measuring cytokines in serum. Thus, serum from naive or infected mice with or without tumors was analyzed by LegendPlex analysis according to the manufacturer's protocol (BioLegend).

[0129] clinical chemistry Induction of liver pathology by LCMV was determined by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Mice were bled on various days after LCMV infection and serum was extracted. Analysis of ALT and AST values ​​was performed at the Zentrallabor, Universitatsklinkum Essen or the Zentralinstitut fur Klinische Chemie und Laboratoriumsdiagnostik, HHU Düsseldorf.

[0130] Analysis of immune cells by flow cytometry To determine the effect of LCMV therapy, immune cells were analyzed by flow cytometry (LSR Fortessa, BD). Platelets were detected by their specific size and granularity. T cells were stained with specific fluorescently labeled antibodies against CD8, CD4, and CD3 (eBioscience). LCMV-specific T cells were stained using tetramer staining (NIH, Tetramer Facility).

[0131] Tumor Growth and Treatment To measure the antitumor effect of LCMV therapy, C57BL / 6 mice (6–12 weeks old) were inoculated with 1 x 10 6 Tumor cells (in 100 μL) were injected subcutaneously. After visible tumor formation, animals were treated with LCMV or control and the mean tumor volume was determined. End-point analysis of tumor burden was performed by measuring the total tumor weight (in g).

[0132] statistical analysis For histograms, means were compared using an unpaired two-sample Student's t test. Tumor growth curves were compared using two-way analysis of variance and Skidak's multiple comparison test. Data are presented as SEM ± mean. Statistical significance level was determined to be p<0.05.

[0133] the study Example 1 C57BL / 6 mice were treated subcutaneously with B16 melanoma cells at 2x10 4 Mice were infected intravenously with PFU of LCMV strains WE or WE-Cl13(L) or control treated (n=5 animals per group). Tumor growth was measured over 7 days. Tumor masses were harvested at completion (d7) (Figure 1).

[0134] This demonstrated that LCMV WE-CL13(L) exhibits increased antitumor efficacy compared to wild-type LCMV strain WE in the non-immunogenic cold B16 melanoma tumor model, an example of a cold tumor model.

[0135] Example 2 C57BL / 6 mice were treated subcutaneously with B16 melanoma cells at 2x10 4 Mice were infected intravenously with PFU of LCMV strain WE or WE-Cl13(L) or were control treated (n=5 animals per group). Mice were sacrificed 7 days after infection. Tumor-resident cytotoxic CD8 + T cells were stained with fluorescently labeled antibodies (CD8 eBioscience) and analyzed by flow cytometry (Figure 2).

[0136] This demonstrated that LCMV strain WE-Cl13(L) enhances T cell infiltration into tumor tissue.

[0137] Example 3 C57BL / 6 mice were treated subcutaneously with B16 melanoma cells at 2x10 4 Mice were infected intravenously with PFU of LCMV strain WE or WE-Cl13(L) or control treated (n=5 animals per group). Mice were sacrificed 7 days after infection. Tumor-resident cytotoxicity, antiviral CD8 + GP33-tetramer positive T cells were analyzed by flow cytometry on day 7 postinfection (Fig. 3 ).

[0138] This demonstrated that the LCMV strain WE-Cl13(L) induced stronger adaptive immune activation than the wild-type LCMV strain WE.

[0139] Example 4 C57BL / 6 mice were treated subcutaneously with MC38 colon cancer cells. Mice were treated with 2x10 4 Mice were infected intravenously with PFU of LCMV strain WE or WE-Cl13(L) or control treated (n=4 animals per group). Tumor growth was observed for 11 days (Figure 4).

[0140] This demonstrated that LCMV strain WE-Cl13(L) exhibited strong tumor growth inhibitory effects in the immunogenic warm MC38 tumor model, which is a model of warm tumors.

[0141] Example 5 C57BL / 6 mice were treated subcutaneously with MC38 colon cancer cells. Mice were treated with 2x10 4 Animals were infected intravenously with PFU of LCMV strain WE or WE-Cl13(L) or control treated (n=4 animals per group). Tumor tissues were dissected 11 days after infection. Tumor-infiltrating T cells (CD3 + ) was stained with a fluorescently labeled antibody (CD3, eBioscience) and measured by flow cytometry (Example 5).

[0142] This demonstrated that LCMV strain WE-Cl13(L) induces strong T cell infiltration into tumors.

[0143] Example 6 C57BL / 6 mice were treated subcutaneously with MC38 cells at 2x10 4 Animals were infected intravenously with PFU of LCMV strain WE or WE-Cl13(L) or were control treated (n=4 animals per group). Tumor tissues were dissected 11 days after infection. Tumor-infiltrating cytotoxic T cells (CD3 + CD8 + ) were stained with fluorescently labeled antibodies (CD3, CD8, eBioscience) and analyzed by flow cytometry (Figure 6).

[0144] This demonstrated that LCMV strain WE-Cl13(L) strongly enhanced cytotoxic T cell infiltration into tumors.

[0145] Example 7 C57BL / 6 mice were treated subcutaneously with MC38 cells at 2x10 4 Mice were infected intravenously with PFU of LCMV strain WE (n=4) or WE-Cl13(L) (n=3) or were control treated (n=4). Tumor tissues were histologically analyzed. Tumor sections (7 μm) were analyzed for LCMV nucleoprotein (LCMV NP, clone VL4) and CD8 + T cells (CD8) were stained with a fluorescently labeled antibody (Figure 7).

[0146] This demonstrated that LCMV strain WE-Cl13(L) enhanced cytotoxic T cell infiltration into tumors independent of the specific local viral presence.

[0147] Example 8 C643, H1975, Ma-Mel-51, SkMM, B16F10, MC57G and HEK293T cells were seeded (100.000 cells / well) and infected with LCMV strains WE, clone 13, WE-Cl13(L) (WE S segment, clone 13 L segment) and clone 13-WE(L) (clone 13 S segment, WE L segment) at MOI=0.1. Cells were cultured for 20 h and stained intracellularly for the presence of LCMV nucleoprotein by fluorescently labeled antibody (NP, clone VL4). The percentage of infected cells from the total cells was analyzed by flow cytometry (Figure 8).

[0148] This demonstrated that the clone 13 L segment was responsible for viral attenuation in all tumor cell lines, but did not significantly reduce infection of the producer cell line HEK293.

[0149] Example 9 C643, Ma-Mel-86a, Ma-Mel-51, 511950, B16F10, Tramp-C2 and JAWSII cells were seeded (100.000 cells / well) and infected with LCMV strains WE and WE-C113(L) at MOI = 0.1. At 24 and 48 hours post-infection, supernatants were harvested and analyzed for LCMV replication by LCMV focus-forming assay (Example 9).

[0150] This demonstrated that the replicative capacity of LCMV strain WE-Cl13(L) was attenuated in tumor cells and antigen-presenting cells (JAWSII).

[0151] Example 10 Primary human neurons, mouse MC57G cells, human lung adenocarcinoma cell line A549 and human thyroid carcinoma cell line FTC133 were seeded (100.000 cells / well) and infected with LCMV WE and WE-Cl13(L) at an MOI of 0.1. Supernatants were harvested 48 hours later and analyzed for LCMV replication by focus formation assay (Figure 10).

[0152] This demonstrated that LCMV strain WE-Cl13(L) exhibits a lack of neurotropism compared to LCMV strain WE, but at the same time exhibits enhanced replication in susceptible tumor cells such as the murine fibrosarcoma cell line MC57G, as well as robust replication in the human cancer cell lines A549 and FTC133.

[0153] Example 11 Bone marrow-derived dendritic cells were infected with LCMV strains WE and WE-Cl 13. Supernatants were collected and analyzed for interferon-alpha protein by a commercially available interferon-alpha ELISA kit (Figure 11).

[0154] This demonstrated that LCMV strain WE-Cl13(L) induces an interferon response similar to that of LCMV WE upon in vitro infection of dendritic cells.

[0155] Example 12 2x10 C57BL / 6 mice 4 Mice were infected intravenously with PFU of LCMV strains Armstrong (Arm, n=3), WE (n=3), WE-Cl13(L) (n=3) or clone 13 (Cl13, n=3) or left untreated (n=12). Blood was collected on days 1 and 3 post-infection. Serum cytokine concentrations of IFNα and IFNβ were analyzed by LegendPlex assay (BioLegend) (Figure 12).

[0156] This demonstrated that the LCMV WE-Cl13(L) reassortant induced reduced levels of interferon in mice upon systemic administration.

[0157] Example 13 2x10 C57BL / 6 mice 4 Mice were infected intravenously with PFU of LCMV strains Armstrong (Arm, n=3), WE (n=3), WE-Cl13(L) (n=3) or clone 13 (Cl13, n=3) or left untreated (n=12). Blood was collected on days 1 and 3 post-infection. Serum cytokine concentrations of IL-6, Il-10, TNFα and IFNγ were analyzed by LegendPlex assay (BioLegend) (Figure 13).

[0158] This demonstrated that LCMV strain WE-CL13(L) induced lower concentrations of Th1 and proinflammatory cytokines in mice.

[0159] Example 14 2x10 wild-type C57BL / 6 or BALB / c mice or the highly susceptible mouse strain FVB / N 6 Mice were infected intravenously with PFU of LCMV strains WE, Cl13 (clone 13), Arm (Armstrong) or WE-Cl13(L) (n=3). Body weights were measured over a 14 day period. Body weights were analyzed as a percentage of body weight on the day of infection (Figure 14).

[0160] This demonstrated that the LCMV reassortant WE-Cl13(L) did not induce lethal pathology in the highly susceptible mouse strain FVB / N compared to the wild-type LCMV strain WE.

[0161] Example 15 2x10 wild-type C57BL / 6 or BALB / c mice or the highly susceptible mouse strain FVB / N 6Mice were infected intravenously with PFU of LCMV strains WE, Cl13 (clone 13), Arm (Armstrong) or WE-Cl13(L) (N=3). Mice were bled on days 3, 6 and 9. Platelets (thrombocytes) were measured in whole blood by flow cytometry. The total number of platelets per microliter of blood was analyzed (Figure 15).

[0162] This demonstrated that LCMV strain WE-C113(L) does not induce thrombocytopenia in highly susceptible FVB / N and other mouse strains.

[0163] Example 16 2x10 wild-type C57BL / 6 or BALB / c mice or the highly susceptible mouse strain FVB / N 6 Mice were infected intravenously with PFU of LCMV strains WE, Cl13 (clone 13), Arm (Armstrong) or WE-Cl13(L) (N=3). Mice were bled on days 3, 6, 9 and 14. Serum levels of alanine aminotransferase (ALT) were analyzed (FIG. 16).

[0164] This demonstrated that LCMV WE-Cl13(L) does not induce liver pathology in highly susceptible FVB / N and other mouse strains.

[0165] Example 17 2x10 wild-type C57BL / 6 or BALB / c mice or the highly susceptible mouse strain FVB / N 6 Mice were infected intravenously with PFU of LCMV strains WE, Cl13 (clone 13), Arm (Armstrong) or WE-Cl13(L) (N=3). Mice were bled on days 3, 6, 9 and 14. Serum levels of aspartate aminotransferase (AST) were analyzed (Figure 17).

[0166] This demonstrated that LCMV WE-Cl13(L) does not induce liver pathology in highly susceptible FVB / N and other mouse strains.

[0167] Example 18 B16 melanoma tumor cells were injected subcutaneously into C57BL / 6 mice. Mice were cultured at 2x10 4 Mice were treated with PFU of LCMV strains WE (n=4) or WE-Cl13(L) (n=5), or control-treated (n=6). Blood was collected 7 days post-infection and serum levels of ALT and AST, as indicators of liver pathology, were analyzed (Figure 18).

[0168] This demonstrated that LCMV strain WE-CL13(L) does not induce liver pathology after systemic administration in the presence of LCMV replication in tumors.

[0169] Example 19 OVA-expressing B16 tumor cells were injected subcutaneously into C57BL / 6 mice. Mice were cultured at 2x10 4 Mice were treated intravenously with PFU of LCMV strains WE or WE-Cl13(L) or control-treated (n=5). Mice were sacrificed 7 days after treatment and spleen, liver, lungs, kidneys and brain were analyzed for replicating LCMV particles by focus-forming assay (FIG. 19).

[0170] This demonstrated that LCMV strain WE-Cl13(L) exhibited significantly reduced replication in healthy organs.

[0171] Example 20 Murine MC38 tumor cells were implanted subcutaneously into C57BL / 6 mice. Mice were then treated with 2x10 4 Tumor tissues were analyzed for LCMV replication by focus formation assay at day 11 post-infection (Figure 20).

[0172] This demonstrated that LCMV strain WE-Cl13(L) replicated equally to LCMV strain WE in tumor tissue.

[0173] Example 21 Primary human neurons, lung adenocarcinoma cell line H1975, gastrointestinal cancer cell line GIST-T1, primary melanoma cell line Ma-Mel-86a, mouse fibrosarcoma MC57G and primary melanoma cell line Ma-Mel-51 were seeded (100.00 cells / well) and infected with LCMV strains WE, WE-Cl13(L) or P52-Cl13(L) at an MOI of 0.1. After 20 hours, cells were stained intracellularly for LCMV nucleoprotein (NP, clone VL4). LCMV-infected cells (LCMV NP + ) was analyzed by flow cytometry (Figure 21).

[0174] This demonstrated that the LCMV reassortant LCMV WE-Cl13(L) and LCMV P52-Cl13(L) do not replicate in human neurons, whereas LCMV strain P52-Cl13(L) exhibits increased infectivity and replication in human and mouse tumor cells.

[0175] Example 22 Primary human neurons, lung adenocarcinoma cell line H1975, gastrointestinal cancer cell line GIST-T1, primary melanoma cell line Ma-Mel-86a, mouse fibrosarcoma MC57G and primary melanoma cell line Ma-Mel-51 were seeded (100.00 cells / well) and infected with LCMV strains WE, WE-Cl13(L) or P52-Cl13(L) at MOI=0.1. After 20 hours, cells were intracellularly stained for LCMV nucleoprotein (NP, clone VL4). The fluorescence intensity of LCMV-infected cells was analyzed by flow cytometry to examine the replication ability in the cells (Figure 22).

[0176] This demonstrated that LCMV strain P52-C113(L) exhibits enhanced replication in human and mouse tumor cells.

[0177] Example 23 Differentiated human myoblasts, human skeletal myoblasts (SkMM), lung adenocarcinoma cell line A549 and mouse fibrosarcoma cell line MC57G were seeded (100.000 cells / well) and infected with LCMV strains WE, WE-Cl13(L), ​​P52-Cl13(L), ​​I181M-Cl13(L) or R185W-Cl13(L) at an MOI of 0.1. After 20 hours, cells were intracellularly stained for LCMV nucleoprotein (NP, clone VL4). LCMV-infected cells (LCMV NP + ) was analyzed by flow cytometry (Figure 23).

[0178] This demonstrated that mutations in the LCMV glycoprotein at positions 181 and 185 enhanced replication and infectivity in human and mouse cancer cells but did not increase tropism for healthy human muscle cells.

[0179] Example 24 Primary human neurons and mouse fibrosarcoma MC57G cells were tested for their LCMV replication capacity. 100.000 cells were seeded per well and infected with LCMV strains WE, WE-C113(L) or P52-C113(L) at MOI=0.1. Supernatants were harvested 48 hours post-infection. Viral titers were analyzed by focus-forming assay (Figure 24).

[0180] This demonstrated that the reassortant LCMV strains WE-Cl13(L) and P52-Cl13(L) show no tropism for healthy human neurons but exhibit enhanced replication in mouse cancer cells compared to the wild-type LCMV strain WE.

[0181] Example 25 C57BL / 6 mice were subcutaneously treated with mouse melanoma B16 cells at 2x10 4 Mice were infected intravenously with PFU of LCMV strains WE, WE-C113(L) or P52-C113(L) or control treated (n=5 animals per group). Tumor growth was measured over 7 days. Tumor burden was measured at completion (d7) (Figure 25).

[0182] This demonstrated that LCMV strains WE-CL13(L) and P52-Cl13(L) exhibited increased antitumor effects compared to the wild-type LCMV strain WE.

[0183] Example 26 C57BL / 6 mice were treated subcutaneously with B16 melanoma cells at 2x10 4 Mice were infected intravenously with PFU of LCMV strains WE, WE-Cl13(L) or P52-Cl13(L) or were control treated (n=5 animals per group). Mice were sacrificed 7 days after infection. Tumor-draining lymph node resident cytotoxic CD8 + T cells were stained with fluorescently labeled anti-CD8 antibody (eBioscience) and analyzed by flow cytometry (Figure 26).

[0184] This demonstrated that LCMV strains WE-CL13(L) and P52-Cl13(L) enhanced cytotoxic T cell accumulation in tumor-draining lymph nodes.

[0185] Example 27 C57BL / 6 mice were treated subcutaneously with B16 melanoma cells at 2x10 4 Mice were infected intravenously with PFU of LCMV strains WE, WE-Cl13(L) or P52-Cl13(L) or control treated (n=5 animals per group). Mice were sacrificed 7 days after infection. Tumor-draining lymph node resident cytotoxicity, antiviral CD8 + GP33-tetramer positive T cells were stained with fluorescently labeled antibodies (eBioscience, NIH) and analyzed by flow cytometry on day 7 post-infection (FIG. 27).

[0186] This demonstrated that LCMV strain P52-Cl13(L) increased the accumulation of virus-specific T cells in tumor-draining lymph nodes.

[0187] Example 28 C57BL / 6 mice were treated subcutaneously with B16 melanoma cells at 2x10 4Mice were infected intravenously with PFU of LCMV strains WE, WE-C113(L) or P52-C113(L) or control treated (n=5 animals per group). Mice were sacrificed 7 days after infection. Tumor-resident cytotoxic CD8+ T cells were stained with fluorescently labeled anti-CD8 antibody (eBioscience) and analyzed by flow cytometry (Figure 28).

[0188] This demonstrated that LCMV strains WE-CL13(L) and P52-Cl13(L) enhanced cytotoxic T cell accumulation in tumors.

[0189] Example 29 C57BL / 6 mice were treated subcutaneously with B16 melanoma cells at 2x10 4 Mice were infected intravenously with PFU of LCMV strains WE, WE-Cl13(L) or P52-Cl13(L) or control treated (n=5 animals per group). Tumor-draining lymph node resident cytotoxicity, antiviral CD8 + GP33-tetramer positive T cells were stained with fluorescently labeled antibodies (eBioscience, NIH) and analyzed by flow cytometry on day 7 post-infection (FIG. 29).

[0190] This demonstrated that treatment with LCMV strain P52-C113(L) strongly enhanced antiviral T cell accumulation in tumor tissue.

[0191] Example 30 C57BL / 6 mice were treated subcutaneously with murine MC38 colon cancer cells. After visible tumor formation, mice were cultured at 2x10 4 Mice were treated intravenously with PFU of LCMV strains WE, WE-C113(L), ​​P52-C113(L) or control (n=4). Tumor volumes were measured up to 11 days post-infection (FIG. 30).

[0192] This demonstrated that LCMV strain P52-C113(L) induced potent tumor growth inhibition.

[0193] Example 31 C57BL / 6 mice were treated subcutaneously with murine MC38 colon cancer cells. After visible tumor formation, mice were cultured at 2x10 4 Mice were treated intravenously with PFU of LCMV strains WE, WE-Cl13(L), ​​P52-Cl13(L) or control (n=4). Tumors were dissected on day 11. T cells were stained with fluorescent anti-CD3 antibody (eBioscience) and analyzed by flow cytometry (Figure 31).

[0194] This demonstrated that LCMV strain P52-Cl13(L) exhibited a clear trend towards enhanced T cell accumulation in tumor tissue compared to LCMV strains WE, WE-Cl13(L) and control treated tumors.

[0195] Example 32 C57BL / 6 mice were treated subcutaneously with murine MC38 colon cancer cells. After visible tumor formation, mice were cultured at 2x10 4 Mice were treated intravenously with PFU of LCMV strains WE, WE-Cl13(L), ​​P52-Cl13(L) or control (n=4). Tumors were dissected on day 11. Helper T cells were stained with fluorescent anti-CD3 and anti-CD4 antibodies (eBioscience) and analyzed by flow cytometry (Figure 32).

[0196] This demonstrated that LCMV strains WE-Cl13(L) and P52-Cl13(L) induced stronger helper T cell infiltration into tumor tissue compared with tumors treated with wild-type LCMV strain WE or control-treated tumors.

[0197] Example 33 C57BL / 6 mice were treated subcutaneously with murine MC38 colon cancer cells. After visible tumor formation, mice were cultured at 2x10 4 Mice were treated intravenously with PFU of LCMV strains WE, WE-Cl13(L), ​​P52-Cl13(L) or control (n=4). Tumors were dissected on day 11. Cytotoxic T cells were stained with fluorescent anti-CD3 and anti-CD8 antibodies (eBioscience) and analyzed by flow cytometry (Figure 33).

[0198] This demonstrated that LCMV strain P52-Cl13(L) enhanced cytotoxic T cell accumulation in tumors compared to control treatment.

[0199] Example 34 C57BL / 6 mice were cultured at 2x10 per mouse. 4 Mice were infected intravenously with PFU of LCMV strains WE, WE-C113(L), ​​P52-C113(L) or left untreated (n=3). Blood was collected on days 2 and 4 post-infection. Serum cytokine levels of IFNα, TNFα and IFNγ were analyzed by LegendPlex assay (BioLegend) (Figure 34).

[0200] This demonstrated that the LCMV reassortant strains WE-Cl13(L) and P52-Cl13(L) induced reduced systemic cytokine levels compared to the wild-type LCMV strain WE and clone 13.

[0201] Example 35 C57BL / 6 mice were subcutaneously treated with B16-Ova melanoma cells at 2x10 4 Mice were infected intravenously with PFU of LCMV strains WE, WE-C113(L) or P52-C113(L) or control treated (n=5 animals per group). Mice were sacrificed 7 days post-infection. Spleen, liver, lung, kidney, brain and tumor tissues were analyzed for LCMV replication by focus formation assay (Figure 35).

[0202] This demonstrated that the LCMV reassortant P52-Cl13(L) exhibited reduced replication in healthy organs with concomitant measurable replication in tumor tissue.

[0203] Example 36 MC38 tumor cells were implanted subcutaneously into C57BL / 6 mice. After visible tumor formation, mice were cultured at 2x10 4Tumors were treated intravenously with PFU of LCMV strains WE, WE-C113(L) or P52-C113(L). Tumors were dissected 11 days after infection and LCMV replication was analyzed by focus formation assay (Figure 36).

[0204] This demonstrated that LCMV strain P52-CL13(L) persisted for shorter periods in mouse tumor tissues compared to strains WE and WE-Cl13(L).

[0205] Example 37 FreeStyle 293-F suspension cells were inoculated with LCMV strains WE-Cl13(L), ​​P52-Cl13(L), ​​or P52-WE(L) at an MOI of 0.001 at 1.5x10 cells per mL. 6 Cells were infected at a density of 1200 x 1000 cells / well. Supernatants were harvested at 12, 24, 30, 34, 40, 48, 72 and 96 hours post-infection. LCMV titers in the supernatants were analyzed by focus formation assay (Figure 37).

[0206] This demonstrated that FreeStyle 293-F cells exhibited enhanced replication of LCMV reassortants WE-CL13(L) and P52-Cl13(L), ​​demonstrating that they represent an ideal producer cell line for the production of LCMV reassortants.

[0207] The embodiments illustratively described herein may suitably be practiced in the absence of any element or limitation not specifically disclosed herein. Thus, the terms and expressions used herein are used for description and not for limitation, and the use of such terms and expressions is not intended to exclude equivalents of the features shown and described or portions thereof, and recognizes that various modifications are possible within the scope of the invention as claimed. Thus, although the present embodiments have been specifically disclosed with preferred embodiments and optional features, it should be understood that modifications and variations thereof may be utilized by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention. Any subgeneric or subgeneric classifications contained in the disclosure of a genus form part of the invention. This includes the description of the genus of the invention with a conditional or negative limitation that removes any subject matter from the genus, whether or not the deleted material is expressly described herein. In addition, when a feature is described in terms of a Markush group, one skilled in the art will recognize that the disclosure is also described thereby in terms of any individual member or subgroup of members of the Markush group.

[0208] Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

[0209] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0210] All publications cited throughout the text of this specification (including all patents, patent applications, scientific articles, manufacturer's specifications, instruction manuals, and the like) are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. In the event that any material incorporated by reference contradicts or is inconsistent with this specification, the present specification will supersede such material.

[0211] Further aspects will become apparent from the following claims.

Claims

1. A viral particle comprising a lymphocytic choriomeningitis virus (LCMV) S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40, wherein the glycoprotein comprises at least one mutated amino acid residue compared to the glycoprotein sequence set forth in SEQ ID NO:

4.

2. A viral particle comprising a lymphocytic choriomeningitis virus (LCMV) S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40, wherein the L protein comprises an amino acid residue other than Lys at position 1079 corresponding to the linear polypeptide sequence of SEQ ID NO:

40.

3. 3. The viral particle of claim 1 or 2, wherein the L protein comprises a non-basic amino acid residue, preferably a neutral hydrophilic amino acid residue, preferably Asn or Gln, preferably Gln, at position 1079 corresponding to the linear polypeptide sequence of SEQ ID NO:

40.

4. 4. The viral particle according to claim 1, wherein the glycoprotein comprises at least one mutated amino acid residue compared to the glycoprotein sequence set forth in SEQ ID NO:4, preferably at least one mutated amino acid residue at position 181 and / or 185 compared to the glycoprotein sequence set forth in SEQ ID NO:4, preferably at least one mutation selected from Arg 185→Trp and Ile 181→Met compared to the glycoprotein sequence set forth in SEQ ID NO:

4.

5. 5. The viral particle of any one of claims 1 to 4, wherein the S segment is derived from LCMV strain WE and the L segment is derived from LCMV strain clone 13.

6. The viral particle of any one of claims 1 to 5, wherein the S segment comprises an open reading frame encoding a nucleoprotein having at least 97% sequence identity to SEQ ID NO:

6.

7. The viral particle of any one of claims 1 to 6, wherein the L segment comprises an open reading frame encoding a Z protein having at least 90% sequence identity to SEQ ID NO:

38.

8. A viral particle comprising a lymphocytic choriomeningitis virus (LCMV) S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and an open reading frame encoding a nucleoprotein having at least 97% sequence identity to SEQ ID NO:6, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40, and an open reading frame encoding a Z protein having at least 90% sequence identity to SEQ ID NO:

38.

9. The viral particle of any one of claims 1 to 8, which is less pathogenic in mice compared to LCMV strain WE.

10. 10. The viral particle of any one of claims 1 to 9, which exhibits increased replication in tumor cells compared to LCMV strain WE.

11. The viral particle of any one of claims 1 to 10, which exhibits reduced replication in healthy organs compared to a viral particle comprising LCMV strain WE and / or the same S segment as said viral particle and the L segment shown in SEQ ID NO:

2.

12. 12. The viral particle according to any one of claims 1 to 11, wherein the S segment comprises a sequence having or preferably being identical to the sequence set forth in SEQ ID NO: 1 or 11, at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98%. Preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity.

13. the L segment has an identical identity to the sequence set forth in SEQ ID NO:21 or 31 to at least about 83%, preferably at least about 84%, preferably at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% 13. The viral particle according to any one of claims 1 to 12, comprising a sequence having or preferably being identical to said sequence, preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity.

14. 14. A viral particle according to any one of claims 1 to 13, having a bipartite genome.

15. 15. The virus particle of any one of claims 1 to 14, which is an arenavirus particle.

16. A host cell comprising an LCMV S segment and an LCMV L segment as defined in any one of claims 1 to 15.

17. (a) an ORF encoding a glycoprotein as defined in any one of claims 1 to 15, an ORF encoding an L protein as defined in any one of claims 1 to 15, an ORF encoding a nucleoprotein as defined in any one of claims 1 to 15, and an ORF encoding a Z protein as defined in any one of claims 1 to 15; and / or (b) an LCMV S segment and an LCMV L segment as defined in any one of claims 1 to 15. A host cell comprising the cDNA of

18. A method for producing a viral particle according to any one of claims 1 to 15, comprising culturing a host cell according to claim 16 or 17 under conditions suitable for viral particle formation.

19. A pharmaceutical composition comprising a viral particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:

40.

20. A pharmaceutical composition for treating cancer, comprising a viral particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO:4, and the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO:40.

Images